Hypertension is a major risk factor for stroke, heart disease and is a cause of chronic kidney disease. Several monogenetic disorders suggest that defects in epithelial sodium channels (ENaC) themselves or their regulation leads to hypertension. Therefore, understanding regulation of ENaC is important as a potential source of hypertension. The sodium flux through ENaC measured as current, INa, across the apical membrane is given as ????? = ???? where I is the current through one open channel, N is the channel density, and Po is the channel open probability. The amount of current can be altered by changing any of these parameters, but in general only N or Po change to alter transport. A lot of attention has been paid to changes in trafficking that change N, but, we feel that changes in Po are also a major mechanism for altering. We hypothesize that ENaC subunits are associated with specific domains of the apical membrane that contain ENaC signaling complexes. These domains have a membrane component which is rich in cholesterol and inositol lipid phosphates. They also have several membrane-associated proteins (MARCKS or MLP-1) that can modify ENaC activity by acting as PIP2- sequestering agents that present PIP2 to ENaC. All of these components are organized through cytoskeletal interactions. The inositol lipid phosphates within the domain anchor the signaling proteins (MARCKS or MLP-1) in the domain, but also interact with and increases the Po of individual ENaC channels. The association of ENaC with the domain, the inositol lipid phosphates, and the signaling complex also stabilizes the functional channels so they have relatively long half-lives. To test these hypotheses, we will: (1) examine binding of PIP2 to ENaC to determine if binding opens the channel or if the binding only promotes a state that has a higher probability of opening; (2) determine if ENaC is present in PIP2-rich lipid domains and determine if the presence of the PIP2- binding proteins, MARCKS or MLP-1, is necessary to maintain ENaC in these lipid domains; (3) examine the regulation of the pMARCKS/MLP-1 and how MARCKS/MLP-1 regulation;(4) examine the proteolytic regulation of MARCKS/MLP-1 by calpains and how the cleaved form of MLP-1 is stabilized; and (5) determine the effect of scaffolding proteins like MAL on the localization and stability of MARCKS and ENaC in the membrane. Determine if MAL is necessary for ENaC insertion into PIP2 rich domains of the apical membrane and, once ENaC is inserted, does MAL stabilize ENaC (and MARCKS) in the specialized domains.